The use of amphipathic maleimides to study membrane-associated proteins

A series of amphiphilic polymethylenecarboxymaleimides has been synthesized for use as sulfhydryl reagents applicable to membrane proteins. Physical properties of the compounds which are relevant to their proposed mode of action have been determined. By comparing rates of reaction in aqueous and aprotic solvents, the compounds have been shown to react exclusively with the thiolate ion. The effects of the reagents on three membrane-associated proteins are reported, and in two cases a comparative study has been made of the effects on the proteins in the absence of membranes. A mechanism is proposed whereby the reagents are anchored at the lipid/water interface by the negatively charged carboxyl group, thus siting the reactive maleimide in a plane whose depth is defined by the length of the reagent. Supporting evidence for this model is provided by the inability of the reagents to traverse membranes, and variation of their inhibitory potency with chain length when the proteins are embedded in the membrane, but not when extracted into solution. As examples of general use of the reagents to probe sulfhydryl groups in membrane proteins, the reagents have been used to (a) determine the depths in the membrane at which two populations of sulfhydryl groups occur in the mitochondrial phosphate transporter; (b) locate a single sulfhydryl associated with the active site ofD--hydroxybutyrate dehydrogenase in the inner mitochondrial membrane; (c) examine sulfhydryl groups in theD-3-glyceraldehyde phosphate dehydrogenase associated with the human red blood cell membrane.     

Sedimentary records of accelerated nutrient loading in Florida lakes

Transfer functions relating trophic state (Carlson's TSI_{chlorophyll-a}) to present day accumulation rate of (1) nutrients, (2) cations, and (3) organic sediment, are computed using Binford's ²¹⁰Pb-dilution method. As computed from surficial sediments of 27 lakes, former trophic states are reconstructed for recent (²¹⁰Pb-dated) sedimentary histories of 14 lakes. Of the three kinds of models potentially available, model (3) (TSI vs. organic accumulation) is the weakest statistically, and may be unduly influenced by exceptional deposition and/or preservation of allochthonous organic matter. At present, however, it is the only model applicable to all 14 of the histories tested. Results are encouraging in that model accurately predicts observed TSI's in several mesotrophic and eutrophic lakes. Clearly significant increases (accelerations) are inferred only for a of the most eutrophic lakes of the set, while the model consistently overpredicts TSI's of the 7 most oligotrophic lakes. As Whitmore's diatom-assemblage index is a better predictor of TSI than is model (3) in the one eutrophic lake in which it has been tested, we expect more persuasive results when models (1) and (2) can be tested within a more complete set of analytical data. We were surprised to find 3 severely disturbed lakes among the 12 that show little or no acceleration in rate of eutrophication in recent decades, but we defer attempts at explanation until former nutrient loading can be tested by model (1).     

A rapid, highly efficient trapping system for the collection of 3-indoleacetic acid from a gas chromatography column.

A technique is described in which the entire effluent from a microcolumn is led directly into the flow-stream of an autoanalyzer and mixed with a fluorogenic substrate in buffer. The result is a continuous trace of the elution profile of enzyme activity on the recorder chart. Good separations of four N-acetyl-β-d-hexosaminidase components of human tissues were achieved in less than 30 min on 5 μl samples from 1% $\text{w}\text{v}$ homogenates.     

Alaska peninsula stable isotope and radioisotope chemistry: a study in temporal and adaptive diversity

Purified bone collagen from a small suite of human remains recovered at three sites on the Alaska Peninsula (Port Moller, Brooks River, and Mink Island) were analyzed for stable carbon and nitrogen isotope chemistry and were accelerator radiocarbon dated. Because samples sizes were small and faunal isotope chemistry lacking, results should be considered preliminary. However, these data indicate that each locality was represented by a somewhat different suite of subsistence strategies and covered a distinct temporal span. Port Moller burials (n = 7) from the Hot Springs site date to a calibrated 2σ range of 3547-1388 BP. Although marine foods clearly made the greatest contribution to these diets, individuals were not as heavily reliant on high-trophic-level marine taxa as eastern Aleutian groups to the west, given their intake of salmon and evidence of caribou hunting. Brooks River burials (n = 9) expressed an inland foraging focus with significant reliance on caribou and spawning salmon and covered a calibrated 2σ range of 1484-381 BP. In contrast, individuals from Mink Island (n = 7), dating to cal. 666-292 BP, were heavily reliant on high-trophic-level marine prey similar to but not as enriched isotopically as the Aleut, perhaps a consequence of limited access to caribou and greater reliance on invertebrates. This pattern suggests that prehistoric economic strategies on the Alaska Peninsula were diverse, characterized by fine-tuned adaptations to local ecological settings, perhaps mediated by ethnic factors and territorial and social pressures.     

Nitric oxide and superoxide dismutase modulate endothelial progenitor cell function in type 2 diabetes mellitus

Background

The renin-angiotensin aldosterone system (RAAS) plays an important role in regulating the blood pressure and the genetic polymorphisms of RAAS genes has been extensively studied in relation to the cardiovascular diseases in various populations with conflicting results. The aim of this study was to determine the association of five genetic polymorphisms (A6G and A20C of angiotensinogen (AGT), MboI of renin, Gly460Trp of aldosterone synthase and Lys173Arg of adducin) of RAAS genes in Malaysian essential hypertensive and type 2 diabetic subjects.

Methods

RAAS gene polymorphisms were determined using mutagenically separated PCR and PCR-RFLP method in a total of 270 subjects consisting of 70 hypertensive subjects without type 2 diabetes mellitus (T2DM), 60 T2DM, 65 hypertensive subjects with T2DM and 75 control subjects.

Results

There was significant difference found in age, body mass index, systolic/diastolic blood pressure, fasting plasma glucose and high density lipoprotein cholesterol levels between the hypertensive subjects with or without T2DM and control subjects. No statistically significant differences between groups were found in the allele frequency and genotype distribution for A20C variant of AGT gene, MboI of renin, Gly460Trp of aldosterone and Lys173Arg of adducin (p > 0.05). However, the results for A6G of AGT gene revealed significant differences in allele and genotype frequencies in essential hypertension with or without T2DM (p < 0.001).

Conclusion

Among the five polymorphisms of RAAS genes only A6G variant of AGT gene was significantly associated in Malaysian essential hypertensive and type 2 diabetic subjects. Therefore, A6G polymorphism of the AGT gene could be a potential genetic marker for increased susceptibility to essential hypertension with or without T2DMin Malaysian subjects.     

Standardization of Misleading Immunoassay Based 25-Hydroxyvitamin D Levels with Liquid Chromatography Tandem-Mass Spectrometry in a Large Cohort Study

Background

The interest in vitamin D measurement has strongly increased in recent years. The best indicator for circulating vitamin D levels is 25-hydroxy-vitamin D (25(OH)D) which is often measured by different immunoassays. We demonstrate problems in comparability of measures by different immunoassays and the need for standardization in the context of a large population-based cohort study.

Methods

25(OH)D was measured with the immunoassays Diasorin Liaison in 2006 in 5,386 women and in the context of another project with IDS-iSYS in 4,199 men in 2009–2010 (when the Diasorin Liaison was no longer available in the version utilized in 2006). Standardization was performed by re-measuring of 25(OH)D levels in 97 men and 97 women with liquid chromatography tandem-mass spectrometry (LC-MS/MS) to obtain linear regression conversion equations.

Results

Applying a 30 nmol/L cut-off value for vitamin D deficiency would have resulted in 48.3% of women and 12.1% of men with vitamin D deficiency ahead of standardization. The large gender difference was strongly attenuated after standardization of the assays with only 15.7% of women and 14.3% of men with vitamin D deficiency. Standardization on average increased the 25(OH)D levels by 10.3 nmol/L in women and decreased 25(OH)D levels by 2.9 nmol/L in men.

Conclusion

The standardization with LC-MS/MS revealed that much of the observed gender difference was only assay-driven and the extremely high proportion of 48.3% vitamin D deficient women proved to be an exaggeration of the old version of the Diasorin-Liaison immunoassay. Standardization of 25(OH)D immunoassay results by LC-MS/MS is recommended to improve their accuracy and comparability, provided the LC-MS/MS method itself is adequately validated and standardized.